Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB, a virulence effector protein secreted by Salmonella typhimurium.

The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonella typhimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6(-/-) mouse embryonic fibroblasts (MEFs) compared with Traf6(+/+) MEFs. However, the ectopic expression of TRAF6 in Traf6(-/-) MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host's E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.

A phycoerythrin isolated from Rhodomonas salina induces apoptosis via ERK/Bak and JNK/Caspase-3 pathway in A549 cells.

Rhodomonas salina, Cryptophyta, Rhodomonas genus, is a valuable source for live feed in aquaculture and for the production of phycoerythrin (PE). In this study, PE was extracted from Rhodomonas salina and characterized as having a molecular weight of approximately 24 kDa, an absorbance at 545 nm, and a purity of up to 6.61 (which meets reagent grade requirements with an OD545/OD280 ratio >4). The effects of PE on anticancer activity and its underlying mechanisms were evaluated to assess the immunomodulatory potential on the human lung cancer A549 cell line. Biochemical assays and western blot analysis were applied to confirm the immune mechanisms. The results showed that after 24 h of exposure to PE, the proliferation of A549 cells was significantly and dose-dependently decreased. PE also caused the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential (MMP). The further results showed that PE can remarkably enhance the protein levels of cleaved caspase-3 and p53. Simultaneously, the BCL-2 family was also affected and had some changes, such as the dramatically enhance of Bim and Bak and the decrease of Bcl-2 level. However, it is interesting to note that there was no apparent alteration in Bax expression during the experiment. Furthermore, the biological mechanism for the potential of PE to induce apoptosis showed that the ERK/Bak and the JNK/caspase-3 signaling pathway were activated. This study provides evidence that the anticancer activity of PE in Rhodomonas salina may have potential for preventing cancer and serving as a novel immunostimulant in the pharmaceutical industry.

Improvement of solubility, gelation and emulsifying properties of myofibrillar protein from mantis shrimp (Oratosquilla oratoria) by phosphorylation modification under low ionic strength of KCl.

The objective of this study was to investigate the mechanisms of phosphates coupled with KCl (at 0, 0.01 and 0.03 M) affecting gel and emulsifying properties of myofibrillar protein (MP) from mantis shrimp. 31P NMR showed that phosphate groups were introduced to MP after treatment with sodium pyrophosphate (SPP) or sodium tripolyphosphate (STPP). The incorporation of phosphates enhanced electronegativity and solubility of MP, leading to exposure of hydrophobic groups, especially in the presence of 0.03 M KCl. These changes resulted in increased gel strength, water-holding capacity and elasticity of MP, mainly due to the improved cross-linking of proteins via ionic interaction, hydrophobic interaction and disulfide bonds. Furthermore, the combination of phosphate and KCl contributed to formation of more stable emulsions stabilized by MP with higher emulsifying activity and smaller droplet size. These results indicated that phosphates and low-dose sodium substitutes synergistically improved the processing properties of muscle food.

Exploration of the Extraperitoneal Approach for Single-Level Anterior Lumbar Interbody Fusion: Imaging, Anatomical and Clinical Research.

Background: To explore extraperitoneal approach as an optimal option for reducing peritoneal disruption at a single-level disc in anterior lumbar interbody fusion (ALIF). Methods: First, abdominal axial CT images obtained from 111 patients were observed to evaluate the distribution of extraperitoneal fat at L2-S1 and measure the lateral distances between the midline and the lateral borders of the rectus and the extraperitoneal fat for each disc level. Second, eight embalmed corpses were dissected along the lateral border of the rectus to expose the peritoneum, which was then separated laterally and medially to evaluate the distribution of fat and peritoneum adhesion. Finally, a total of 58 patients were selected for ALIF. For L2-L4 discs and L4-S1, the pararectus approach and the paramedian approach were utilized, respectively. Results: Extraperitoneal fat was observed behind the rectus at the L5-S1 and the lateral distance between the fat and midline and the lateral border of the rectus gradually decreased on both sides of L2-5. On the cranial side of the arcuate line, it was easier to separate the peritoneum outward along the lateral edge of the rectus. When bluntly dissected medially, the peritoneum was closely adhered to abdominal wall. No complications such as peritoneal damage, retroperitoneal hematoma and neurological complications occurred in 58 patients undergoing the aforementioned surgical methods. Conclusions: For L4-S1, the paramedian approach is the optimal technique to expose the disc, whereas the pararectus approach is the feasible surgical method at L2-4.

Influence of sodium tripolyphosphate coupled with (-)-epigallocatechin on the in vitro digestibility and emulsion gel properties of myofibrillar protein under oxidative stress.

The objective of this study was to investigate the effect of (-)-epigallocatechin (EGC; at 0, 10, and 100 µmol g-1 protein) coupled with sodium tripolyphosphate (STP) on the in vitro digestibility and emulsion gel properties of myofibrillar protein (MP) under oxidative stress. The addition of both EGC and STP inhibited protein carbonyl formation but promoted the loss of thiol and free amine groups. Combined with the results of tryptophan fluorescence, surface hydrophobicity, electrophoresis, and solubility, the presence of STP enhanced the covalent reactions between the quinone of EGC and the thiols and free amines of MP. The combination of EGC at 10 µmol g-1 and STP increased the protein digestion rate in the gastric tract and contributed to an improved emulsion gel structure with higher gel elasticity, strength, water-holding capacity, and oxidative stability. This improvement could be attributed to the moderation of MP-EGC cross-linking, which was homogeneously formed among the adsorbed and/or unadsorbed proteins. Thus, oil droplets adhered better to the gel matrix. However, EGC at 100 µmol g-1 coupled with STP led to the formation of excessive non-disulfide covalent bonds, which aggravated the aggregation of MP. This ultimately reduced the protein digestibility and the nutritional value, caused the coalescence of oil droplets as well as the collapse of the gel structure, and thus, an overall decrease in the gel properties and oxidative stability. These results indicated that the enhanced oxidative stability and gelling capacity of MP without nutrition deterioration can be attained through tripolyphosphate coupled with lower doses of EGC.

Site-specific incorporation of sodium tripolyphosphate into myofibrillar protein from mantis shrimp (Oratosquilla oratoria) promotes protein crosslinking and gel network formation.

Formation of protein gels in processed muscle foods is one of the most important functionalities. To explore the mechanisms responsible for affecting gel properties of muscle proteins by phosphates, myofibrillar protein (MP) from mantis shrimp (Oratosquilla oratoria) was treated with sodium tripolyphosphate at three pH values (7.0, 8.0, and 9.0). FTIR and UPLC-MS/MS firstly confirmed that phosphate groups were introduced to MP through COP bonds via serine and threonine residues. The incorporation of STP caused increased electronegativity and solubility, more stable α-helix secondary conformation, and reduced tryptophan fluorescence intensity of MP, especially at pH 8.0 and 9.0. These changes led to a finer, ordered and denser three-dimensional gel network microstructure with higher gel strength and elasticity, and water-holding capacity. This study demonstrated that the introduction of phosphate groups could increase negatively charged residues in MP, enhance the crosslinks of proteins through ionic interaction, and promote gel network formation.

Phosphorylation modification of myofibrillar proteins by sodium pyrophosphate affects emulsion gel formation and oxidative stability under different pH conditions.

Formation of a gel matrix, involving interactions between proteins, lipids, and water, plays an essential role in the textural properties of processed meats. This study investigated the effects of sodium pyrophosphate (SPP) on the textural properties and oxidative stability of myofibrillar protein (MP)-stabilized emulsion gels under different pH conditions (5.0-9.0). The SPP-modified MP emulsion gels showed an improved elasticity, strength, water-holding capacity, and oxidative stability at pH 6.0 and 7.0. This improvement should be mainly attributed to the enhanced protein-protein crosslinks via ionic interaction between phosphate groups and -NH3+ of amino acids, which were homogeneously formed among adsorbed and/or unadsorbed proteins, entrapping fractions of MPs (myosin heavy chain, actin, and troponin T) within the network. Therefore, the oil droplets were better adherent to the gel matrix. Nevertheless, increased electrostatic repulsion between protein molecules due to excessive phosphates attached to MPs at pH 8.0 and 9.0, as well as protein precipitation at pH 5.0, caused the collapse of the MP-stabilized emulsion gel structure, and thus, overall decreased the gel properties and oxidative stability. LC-MS/MS results confirmed that phosphate groups were successfully introduced to MPs through C-O-P bonds at pH 6.0, and the phosphorylation sites were found to be on serine residues (Ser14, Ser79, Ser96, Ser148, Ser2427, and Ser5272), threonine residues (Thr118 and Thr926), and tyrosine residues (Tyr215 and Tyr425). The results provided a new aspect for better understanding the effect of polyphosphates in meat protein/oil composite systems.

Pharmacokinetic Study of Swertisin by HPLC-MS/MS After Intravenous Administration in Rats.

A sensitive and reliable high-performance liquid chromatography with tandem mass spectrometry was developed and validated for the quantification of swertisin in rat plasma. The samples were prepared with methanol by protein precipitation. Swertisin was separated by using an Agilent Poroshell 120 EC-C18 column (4.6 mm × 50 mm, 2.7 µm) with the mobile phase consisted of acetonitrile and water containing 0.1% formic acid running at a flow rate of 0.3 mL/min for 5 min. The analytes were detected with the multiple reaction monitoring in the negative electrospray ionization source for quantitative response of swertisin [M-H]- (445.1→281.7) and puerarin (internal standard) [M-H]- (415.1→295.0). Precision (relative standard deviation, RSD%) of the inter-day and the intra-day was <9.89% and 11.34% while accuracy (relative error, RE%) ranged from -6.01% to 4.92% and -3.97% to 4.39%, respectively. Interestingly, the expression of the γ-aminobutyric acid (GABA) receptor subunits (GABAAα1, GABAAα5 and GABAAß1) mRNAs was enhanced in the rat hippocampal neurons treated with swertisin as analyzed by real-time polymerase chain reaction. Moreover, the method was successfully applied to determine the pharmacokinetic of swertisin in rats after tail vein injection with 4.0 mg/kg of the compound. Our results provide useful pharmacokinetic information for swertisin in vivo and suggest that the sedative function of this compound may be through inducing the expression of the GABAA receptor subunits.

Degradation Kinetics of 6‴-p-Coumaroylspinosin and Identification of Its Metabolites by Rat Intestinal Flora.

6‴-p-Coumaroylspinosin (P-CS), a bioactive flavonoid, is typically extracted from Semen Ziziphi Spinosae (SZS). In this study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to determine P-CS for investigating the degradation characteristics of P-CS incubated with rat feces. The results showed that P-CS degraded rapidly and the degradation speeds varied depending upon the P-CS concentrations (3, 15, and 30 µg/mL). The degradation of P-CS processes follow first-order kinetics. On the basis of the mass spectrometry (MS) spectrum mode of the product ions, two main metabolites of P-CS were identified. Swertisin was the main metabolite at 3 and 15 µg/mL, while spinosin was produced when the P-CS concentration was 30 µg/mL. Spinosin and swertisin could improve mRNA transcription levels of glutamate receptor K1, K2, and K3 (GluK1, GluK2, and GluK3) subunits in rat hippocampal neurons. In addition, they showed an obvious synergistic effect in this respect. Collectively, the results can be used to explain the metabolic and pharmacological mechanisms of P-CS.

Garcinol from Garcinia indica Downregulates Cancer Stem-like Cell Biomarker ALDH1A1 in Nonsmall Cell Lung Cancer A549 Cells through DDIT3 Activation.

Nonsmall cell lung cancer (NSCLC) is the predominant type of lung cancer. Patients with NSCLC show high mortality rates because of failure to clean up cancer stem cells (CSCs). The anticancer activity of phytochemical garcinol has been identified in various cancer cell models. However, the effect of garcinol on NSCLC cell lines is still lacking. Of the NSCLC cell lines we tested, A549 cells were the most sensitive to garcinol. Interestingly, Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) was preferentially expressed in A549 cells and downregulated by the addition of garcinol. We also found that garcinol enriched DNA damage-inducible transcript 3 (DDIT3) and then altered DDIT3-CCAAT-enhancer-binding proteins beta (C/EBPß) interaction resulting in a decreased binding of C/EBPß to the endogenous ALDH1A1 promoter. Furthermore, garcinol's inhibition of ALDH1A1 was identified in a xenograft mice model. Garcinol repressed ALDH1A1 transcription in A549 cells through alterations in the interaction between DDIT3 and C/EBPß. Garcinol could be a potential dietary phytochemical candidate for NSCLCs patients whose tumors harbored high ALDH1A1 expression.

The retrogradation properties of glutinous rice and buckwheat starches as observed with FT-IR, 13C NMR and DSC.

The experiment was conducted to study the retrogradation properties of glutinous rice and buckwheat starch with wavelengths of maximum absorbance, FT-IR, (13)C NMR, and DSC. The results show that the starches in retrograded glutinous rice starch and glutinous rice amylopectin could not form double helix. The IR results show that protein inhabits in glutinous rice and maize starches in a different way and appearance of C-H symmetric stretching vibration at 2852 cm(-1) in starch might be appearance of protein. Retrogradation untied the protein in glutinous amylopectin. Enthalpies of sweet potato and maize granules are higher than those of their retrograded starches. The (13)C NMR results show that retrogradation of those two starches leads to presence of ß-anomers and retrogradation might decompose lipids in glutinous rice amylopectin into small molecules. Glutinous rice starch was more inclined to retrogradation than buckwheat starch. The DSC results show that the second peak temperatures for retrograded glutinous rice and buckwheat starches should be assigned to protein. The SEM results show that an obvious layer structure exists in retrograded glutinous rice amylopectin.

Degradation kinetics of jujuboside b by rat intestinal flora in vitro with an RRLC-MS-MS method.

Jujuboside B (JuB) is a main bioactive saponin constituent of Ziziphi Spinosae Semen, which is a traditional herb for the treatment of insomnia and anxiety. However, the detailed metabolic mechanism of JuB is poorly understood. In this study, a novel method of rapid resolution liquid chromatography-triple quadrupole mass spectrometry was developed and validated for the analysis of JuB. With the method, the degradation kinetics of JuB by rat intestinal flora in vitro was investigated. The analysis was performed with an Agilent Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 µm) column and an aqueous mobile phase (containing 0.1% formic acid) modified by methanol. The analyte was measured by multiple reaction-monitoring (MRM) modes with m/z 1043.3 → m/z 749.2. This method was validated with perfect accuracy, precision and limit of quantitation. It showed that jujuboside B (JuB) degradation started slowly as incubation with rat feces. The rate constant was correlated greatly with the concentration of sample solutions. Furthermore, some metabolites were elucidated with their chromatographic behavior and typical fragment ions. The results might help better interpret the metabolic and pharmacological mechanism of JuB.

Pro-apoptotic effects of the novel tangeretin derivate 5-acetyl-6,7,8,4'-tetramethylnortangeretin on MCF-7 breast cancer cells.

Citrus polymethoxyflavone tangeretin (5,6,7,8,4'-pentamethoxyflavone, TAN) displays multiple biological activities, but previous reports showed that TAN failed to induce MCF-7 human breast cancer cells apoptosis. Herein, we prepared 5-acetyl-6,7,8,4'-tetramethylnortangeretin (5-ATAN), and evaluated its cytotoxicity on MCF-7 cells. 5-ATAN revealed stronger cytotoxicity than that of parent TAN in the growth inhibition of MCF-7 cells. 5-ATAN induced apoptosis via both caspase-independent and -dependent pathways, in which 5-ATAN induced the translocation of apoptosis inducing factor and phosphorylation of H2AX as well as poly (ADP-ribose) polymerase cleavage, caspase-3 activation. However, 5-ATAN did not affect extrinsic markers caspase-8, BID, and FADD. Further, 5-ATAN induced the loss of mitochondrial membrane potential (Δψm) by regulating the Bax/Bcl-2 ratio. Loss of Δψm led to the mitochondrial release of cytochrome c which triggered activation of caspase-9. In conclusion, these data indicate that 5-ATAN plays pro-apoptotic cytotoxic roles in MCF-7 cells through both caspase-dependent intrinsic apoptosis and caspase-independent apoptosis pathways.

A possible structure of retrograded maize starch speculated by UV and IR spectra of it and its components.

"Retrogradation" has been used to describe the changes that occur in starch after gelatinization, from an initially amorphous state to a more ordered or crystalline state, which has a significant impact on starch application in food, textiles and materials fields. But mechanism of starch retrogradation is still unclear until now and there is no breakthrough in this area. Here we are speculating a possible structure of retrograded maize starch by UV (binding with iodine) and IR spectra of it and its compositions. We speculate that nucleation of retrograded starch origins from combination of reducing end of amylopectin and non-reducing end of amylose, and retrogradation terminates at combining of non-reducing end of amylopectin and reducing end of amylose. The chain length of resistant digestion retrograded starch should be nearly same. The hydroxyl associated with sixth carbon atoms of glucan must form hydrogen bond with other hydroxyl of starch.

Retrograded starches as potential anodes in lithium-ion rechargeable batteries.

Retrograded starch is a crystal formed by starch molecules with hydrogen bonds. Many literatures have reported its physicochemical character, but its crystal structure is so far unclear. As we isolate amylose and amylopectin from retrograded maize, sweet potato and potato starches in 4.0 M KOH solutions and make them retrograde alone in neutral solution (adjusted by HCl) to form crystal, a new phenomenon appears, crystals of KCl do not appear in retrograded potato amylose, potato amylopectin, and maize amylose, indicating that those crystals may absorb K⁺ and (or) Cl⁻, and those ions probably act with aldehyde of starch or hydroxy of fatty acid attached in starch, such characteristic may make retrograded starches replace graphite as anode with high-capacity in lithium-ion rechargeable batteries.